Hepatitis C virus (HCV) is one of the most widespread infectious diseases in the world. About 170 million people are infected with HCV worldwide with a yearly incidence of 3-4 million. While the acute phase of infection is mostly asymptomatic, the majority of acutely infected individuals develop chronic hepatitis and is at increased risk of developing liver cirrhosis and hepatocellular carcinoma. Thus, HCV infection is a major contributor to end-stage liver disease and in developed countries to liver transplantation.
HCV is a small, enveloped virus classified as a member of the Flaviviridae family. Its genome consists of a 9.6 kb single stranded RNA of positive polarity composed of 5′ and 3′ untranslated regions (UTRs) and one long open reading frame (ORF) encoding a polyprotein, which is co- and posttranslationally cleaved and thus yields the structural (Core, E1, E2), p7 and nonstructural (NS2, NS3, NS4A, NS4B, NS5A, NS5B) proteins.
HCV isolates from around the world exhibit significant genetic heterogeneity. At least 7 major HCV genotypes (genotypes 1-7) have been identified, which differ by 31-33% at the nucleotide level. In addition, there are numerous subtypes (a, b, c, etc.).
Since its discovery in 1989, research on HCV has been hampered by the lack of appropriate cell culture systems allowing for research on the complete viral life cycle as well as new therapeutics and vaccines.
In 2001, a genotype 2a isolate (JFH1) was described (Kato et al., 2001), which yielded high RNA titers in the replicon system without adaptive mutations (Kato et al., 2003).
A major breakthrough occurred in 2005, when formation of infectious viral particles was reported after transfection of RNA transcripts from the JFH1 full-length consensus cDNA clone into Huh7 cells (Wakita et al., 2005) (Zhong et al., 2005).
At the same time, Lindenbach et al. demonstrated that the intragenotypic 2a/2a recombinant genome (J6/JFH1), in which the structural genes (C, E1, E2), p7 and NS2 of JFH1 were replaced by the respective genes of clone J6CF, produced infectious viral particles in Huh7.5 cells (a cell line derived from bulk Huh7 cells) with an accelerated kinetic (Lindenbach et al., 2005). Cell culture derived J6/JFH viruses were apparently fully viable in vivo. Recently the inventors of the present invention developed robust JFH1-based cell culture systems with genotype specific C-NS2 for HCV genotype 1a, 1b, 2b, 3a, 4a, 5a, 6a and 7a.
A part of the structural gene E2, which is present in all of these genotypes, is the Hypervariable Region 1 (HVR1). HVR1 is generally defined as the N-terminal 26-27 amino acids (aa) of the HCV protein E2 and is marked by the highest variability in the entire HCV genome—even higher than that of the other two HCV hypervariable regions: HVR2 and HVR3.
The region is easy to recognize in spite of the high variability due to several conserved residues within the sequence. It has previously been demonstrated that specific targeting of HVR1 by the adaptive immune system of the host likely causes the high variability of HVR1 (Manzin et al. 2000 and Ray et al. 1999).
The inferred immunogenic properties of HVR1, together with the fact that HVR1 specific antibodies are persistently found in patients chronically infected with HCV (Cerino et al. 1997), suggests that an HVR1 specific immune response occurs during an HCV infection, but does not in itself allow clearance of the viral infection.
It has been suggested that HVR1 acts as an immunological decoy by drawing the attention of the immune system away from less immunogenic, but ultimately more effective epitopes outside of HVR1 (Mondelli et al. 2001). This hypothesis is based on the assumption that HVR1 is not crucial for virus-host interactions since it is difficult to imagine how immune responses targeting HVR1 would not interfere with such interactions.
Thus, the proposed interaction of HVR1 with the HCV receptor Scavenger Receptor class B type I (SR-BI) (Scarselli et al. 2002) made the “immunological decoy” hypothesis less likely.
Steinmann et al. have used the HCV intragenotypic recombinant virus, Jc1 (2a/2a) showing that in this specific case HVR1 deletion was tolerated (Steinmann et al., 2007). Like the J6/JFH (2a/2a) virus used by the inventors the Jc1 (2a/2a) virus consists of J6 and JFH1 sequence that has been spliced, but the genotype-junction is at a different location in the HCV genome. As will be shown in the detailed description, viability of HVR1 deleted virus depends greatly on the virus isolates and as such the outcome of HVR1 deletion from J6/JFH could not have been predicted by these earlier observations.